Biological Constituents of Aged Garlic Extract as Biomarker

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Biological Constituents of Aged Garlic Extract as Biomarker

Seung-Taek Yang*

Department of Food Science and Biotechnology, Kyungsung University, Busan 608-736, Korea

Received December 12, 2008 /Accepted January 23, 2009

Garlic (Allium sativum) are an agronomically important genus because of their sulfur flavour components. The majority of the volatiles flavour principles are generated through the enzymatic hy- drolysis of the non-volatile organosulfur compounds. However, these compounds may be possible sources of new novel bioactive and therapeutic principles. Garlic has strong antioxidant activity, and epidemiological studies support the fact that diets rich of garlic may prevent some of the chronic diseases. The health cares of garlic likely arise from a wide variety of components, which may work synergistically. The chemical changes of garlic composition makes it plausible that a variation in proc- essing can lead to acquisition of differential chemical compositions of garlic products. Especially high- ly unstable allicin can easily disappear during processing and are quickly transformed into a various organosulfur compounds. Various supplements of garlic, particularly aged garlic extract (AGE), are known to possess a promising antioxidant potential and are effective in prevention of chronic diseases because of the bioactive constituents. Although all of active ingredients of AGE are not elucidated, water-soluble components of AGE, including S-allylcysteine, S-allylmercaptane, steroid saponins, tetra- hydro-β-carboline derivatives, and fructosyl-arginine, appears to be associated with the pharmaco- logical effects of AGE. Consequently, the allicin free garlic components such as S-allylcysteine, S-allyl- mercaptane, steroid saponins, tetrahydro-β-carboline derivatives, and fructosyl-arginine can be appli- cable to standardization of the quality of commercial garlic products. This review provides an insight into garlic's biomarkers and presents evidence that they may either prevent or delay chronic disease associated with aging.

Key words : Aged garlic extract, organosulfur compound, biological constituents, biomarker

*Corresponding author

*Tel：+82-51-620-4715, Fax：+82-51-622-4986

*E-mail : [email protected]

Introduction

Garlic (Allium sativum) has been used for centuries both as a flavoring and therapeutic agent for the treatments of human diseases and disorders [25]. Although garlic have been using for remedies and food for more than a thousand year, most people used garlic based on their experiences without any knowledge about relationships between bio- logical activities and constituents of aged garlic [23].

Epidemiological, and preclinical studies have shown the close relation between dietary habits including garlic intake, and occurrence of diseases. Garlic has been investigated ex- tensively for health benefits resulting in more than 2,000 publications over the last decade and it is considered one of the best disease preventive foods, based on its varied ef- fects [3].

Many favorable experimental and clinical effects of the consumption of garlic products, including garlic extract,

have been reported. These biological responses include re- duction of risk factor for cancer [22] and cardiovascular dis- eases [29], a stimulation of immune function [24], restoration of physical strength [6], resistance to various stresses [9], en- hanced foreign compound detoxication [1], potential antiag- ing effects [14] and heavy metal chelation [37].

Because of the intense interest in garlic, various commer- cial preparation have become available over the years. These include garlic powder, tablets, oil-macerated garlic, oil of steam-distilled garlic, ether-extracted oil of garlic and etc.

In fact, in some products, garlic is soaked, or extracted with

water, alcohol, wine or vinegar before use. It has been be-

lieved that the medical and beneficial properties may be at-

tributed to specific constituents found in garlic and its ex-

tracts, and many studies suggest that organosulfur com-

pounds are responsible for the biological activities. Another

such preparation is aged garlic extract (AGE) which is pre-

pared by a unique but natural long-term process of aging

extraction for 1 month at 60～70

^o

C. This process causes con-

siderable loss of allicin and increases the concentration of

other new component. Garlic products, including enteric

coated products, revealed that no allicin was found in blood

- Review -

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after oral ingestion. Allicin disappeared very rapidly in stomach.

Interestingly, it is therefore not surprising that the compo- sition and quantity of aged garlic extract can produce other compounds, given such chemical diversity as new sources of biactive compounds [19]. It does not explain the cause of the inconsistency, because, as shown in many documents, allicin potential is not a correct biomarker for controlling the quality of garlic supplements [19]. Standardization of aged garlic extract is a key to delivering consistent quality and biological activity of garlic products. Therefore, in the current view, sulfur compounds generate from garlic as bio- markers and these standard biomarkers are very important for ensuring consistent effects.

Chemical changes of garlic

Garlic is famous for its undesirable odor, arising from alli- cin and other oil-soluble sulfur components. The major com- ponent of garlic is water (65%), and the bulk of the dry weight is composed of fructose containing glucides, fol- lowed by sulfur compounds. Major compounds present in garlic are 97% water-soluble constituents with small amount of 0.7～0.15% oil soluble compounds. Garlic contains unique organosulfur compound, which provides its characteristic flavor and odor, and most of its effective biological activities.

The strong odor of fresh garlic and its ability to generate unpleasant gastric side effects have caused many to favor dietary garlic supplements as an optimal choice for increas- ing garlic intake [20]. In fact, over 90% of investigations on garlics, active principles have focused on the sulfur com- pounds of which comprises 85% of alliin and two main γ- glutamylcysteines [38]. Alliin considered the parent sub- stance of the therapeutically active sulfur components of garlic. Once garlic is cut or crushed, compounds in the intact garlic are converted into hundreds of organosulfur com- pounds in a short time. When garlic is crushed, cut or chewed, alliin is exposed to the enzyme allinase and thio- sulfinated allicin is formed [29]. According to the clinical study, theoretical amounts of allicin delivered to the in- testinal tract from enteric-coated garlic powder still do not reach the blood stream. Even whether the garlic products contains allicin or not, allicin is not detected in the blood stream (Fig. 1). Therefore, allicin is not bioavailable and can- not reach target organs via circulation.

When allicin itself was kept at 20

^o

C for 10 hr, it decom- posed to diallyl disulfide (DADS), diallyl sulfide (DAS),

Fig. 1. Fate of allicin. No allicin is found in the blood after oral consumption of 90 mg allicin [23]. According to the clinical study, theoretical amounts of allicin delivered to the intestinal tract from enteric-coated garlic powder still do not reach the blood stream. Even if any allicin were to reach the liver from the intestinal tract, it would be transformed in liver tissue. Whether the garlic preparation/product contains allicin or not, allicin is not detected in the bloodstream in the body. Therefore, allicin is not bioavailable and cannot reach targed organs via circulation.

diallyl trisulfide (DATS) and sulfur dioxide [6] (Fig. 2).

One of the main sulfur containing compounds in intact garlic are converted into S-allyl -cysteine (SAC) through an enzymatic transformation with γ-glutamyltranspeptidase when garlic is extracted with water. Although freshly crush- ed, garlic may contain limited amounts of allicin, while no

Fig. 2. Chemical change in garlic. Intact garlic bulbs contain high amounts of γ-glutamylcysteines. These reserve compounds can be hydrolyzed and oxidized to form alliin [41], which accumulates naturally during storage of garlic bulbs at cool temperatures. Allicin and other thiosulfinates instantly decompose to other compounds, such as diallyl sulfide (DAS), diallyl disulfide (DADS) and dia- llyltrisulfide (DATS), dithiins and ajoene. At the same time, γ-glutamylcysteines are converted to S-allylcysteine (SAC) via a pathway other than the alliin/allicin pathway. SAC contributes heavily to the health benefits of garlic.

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commercially available processed garlic products contain allicin. These results indicate that allicin does not contribute to the in vivo effects of garlic. Allicin and allicin potential is not a correct biological biomarker for controlling the qual- ity of garlic product [5,23]. As mentioned above, garlic changes it characteristics because of the complexity of its intrinsic chemistry, which are very important for biological effects. However, some studies shed doubt on the efficacies of garlic, and careful examination of such research can help clarify the processing of garlic. For example, aged garlic extract. Although many garlic preparations are commercially available, confusion remains because of the inconsistency of clinical study results and the lack of scientific studies on in- dividual products.

Therefore, this article focuses on biological properties of main constituents derived from aged garlic extract, which might have an important role in the benefits of garlic to hu- man health.

Commercially available products of aged garlic extracts (AGE)

Garlic supplement products have experienced increasing popularity in the last decade. Market research indicates that garlic products were the most popular herbal supplement in the single herb category. There are many brands of garlic products that provide a convenient way to intake benefits of garlic.

Over the years different garlic products have been studied for their prevention and treatment of various human dis- eases with clinical trials. The garlic products that have been studied included raw garlic, garlic powder, steam distilled garlic oil, macerated garlic oil, and aged garlic extract (AGE) (Table 1).

All these products differ in their composition, which makes comparing them difficult. It is well known that ex- traction product such as AGE increases potency generally and safely, and more effectively compared with raw garlic, garlic powder, macerated garlic oil, or others. Although there is no standard intake of garlic, some researcher sug- gested that daily intake of 1～2 cloves garlic or 1～4 g of intact garlic may have health benefits. The daily dose of garlic powder is 900 mg. However, recommendation is not substantiated with clinical studies. AGE intakes ranging from 1 to 7.2 g have been used with success. Previously, no severe toxic side effects were reported in these clinical studies [9]. AGE has been examined in clinical tests and

Table 1. Garlic products on the market

Type of product Main compounds and characteristics

Garlic essential oil

Only 1% of Oil-soluble sulfur compounds (DAS, DADS, etc.) in 99% vegetable oil No water-soluble fraction

No allicin

Not well-standardized No safety data Garlic oil

macerate Oil

Soluble sulfur compounds and alliin No allicin

Not well-standardized No safety data

Garlic powder

Alliin and a small amount of oil-soluble sulfur compounds

No allicin

Not well-standardized

Results on cholesterol is not consistent.

No safety data

Aged garlic extract (AGE)

Mainly water-soluble compounds (SAC, SAMC, saponins, etc.) Standardized with SAC

Small amount of oil-soluble sulfur compounds Various beneficial effects

Well-established safety

Heavily researched (400 + papers)

Allicin is a highly unstable and reactive compound that rapidly decomposes to other compounds. For this reason no garlic product on the market contains a dete-ctable amount of allicin (<1 μg/g) [13].

show no contraindications with several medications includ- ing cholesterol lowering agent such as statin, aspirin, dox- ysorubin etc. AGE is prepared of eliminated toxicity and other strong unpleasant odors of garlic.

Bioavailability of aged garlic extract

Many favorable experiments and clinical studies on the consumption of garlic products, especially of aged garlic ex- tract (AGE), show a wide variety of biological attributes to it. AGE also has more effects against cardiovascular disease [9,30], cholesterol lowering effect [42], antioxidant [15] and blood coagulability [35]. These additional biological effects may be due to conversion compounds that are formed dur- ing long term extraction process, called the aging process.

AGE is an odorless product resulting from prolonged aging

or extraction of fresh garlic at high temperature and

humidity. It is highly bioavailable and has biological activity

in vitro and in vivo in both animals and humans [7,10]. AGE

contains water soluble allyl amino acid derivatives, which

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account for most of its organosulfur content, soluble allyl sulfides, and flavonoids, saponins, essential macro and micronutrients.

The major unique organosulfur compounds in AGE are water soluble S-allylcysteine (SAC), and S-allylmercaptocys- teine (SAMC), steroid saponin, fructosyl arginine, a Maillard reaction product [39]. In addition, for tetrahydro-β-carboline derivatives which possess hydrogen peroxide, scavenging activity have recently identified in AGE [18]. Among many garlic products, AGE, demonstrating the benefits of the ag- ing process, can eliminate these undesirable compounds, and water soluble sulfur compounds effectively increase the desirable compounds such as SAC for human benefits [31,32] (Fig. 3).

Another such preparation is aged garlic extract (AGE), which is processed by one month under the conditions of 80

^o

C temperature with 70% humidity. This black garlic is extract with water and then filtered under concentrated re- duced pressure at low temperature and is marketed in both dry and liquid forms. This manufacture process causes con- siderable loss of allicin and increases the concentration of other new sulfur-base and water soluble compounds. The major compounds such as polyphenol, S-allylcysteine (SAC), S-allylmercaptocysteine (SAMC), fructosyl arginine (FA), and steroid saponin is used to standardize AGE [23,31].

Several clinical and publications reports that neither aged garlic extract nor dehydrated garlic powder for human benefits. These research data have caused serious confusion in academic and in the public health, because alliin and alli- cin potential is not correct biomarker for showing the quality of garlic products. As mentioned above, garlic changes it properties because of the complexity of its chemistry, during aging process, and standardization biomarker compound are

Fig. 3. SOD-like activity, hydrogen peroxide scavenging activity and total polyphenol content of garlic extract [4].

Hydrogen peroxide scavenging activity is expressed as the % of remaining hydrogen peroxide concentration in which the concentration of the solvent control (80%

EtOH alone) is regarded as 100%.

very useful for consistent activities. Therefore, stand- ardization is a key to delivering parameters of biological ef- fects of garlic products to consumers.

S-allylcysteine (SAC)

SAC content in the intact garlic is a small amount of 20

∼30 μg/g fresh weight, however, SAC is increased in a aging/extraction procedure through hydrolysis of γ-glu- tamyl-S-allylcysteine which exists in raw garlic as a pre- cursor of SAC [35].

Studies on the pharmacokinetics of SAC in a number of animal species show SAC is easily absorbed from the gastro- intestinal tract distributed in plasma, liver and other organs with a bioavailability of 98% in rat [6,17]. Recently, SAC ma- jor a sulfur-containing amino acid compound has been re- ported to have antioxidant activity, anticancer promoting ef- fect, and hepatopathic activity [13,14]. N-acetyl SAC has been identified as a metabolite of SAC in the urine of dogs and humans. These indicated that SAC could be transformed by N-acetyltransferase.

SAC is a very stable compound, although, numberous sul- fur containing compounds in garlic transformed/ decom- posed compounds appear after cleavage of the C-S bond fresh or processed garlic. However, SAC in AGE would be absorbed without any decomposition from changes in gas- trointestinal pH after administration [35]. The pharmacoki- netic study in human was performed by oral administration of garlic product, which contains. show the concentration of SAC in plasma after oral administration of garlic supple- ment by health volunteers (Fig. 4).

Fig. 4. SAC content in human volunteers orally consuming garlic supplement containing SAC [28]. Volunteer F: age, 46; sex, male; body weight, 64 kg; SAC consumed, 0.82 mg. Volunteer H: age, 38; sex, male; body weight, 63 kg;

SAC consumed, 0.67 mg. Volunteer I: age, 45 ; sex, male;

body weight, 65 kg; SAC consumed, 0.67 mg.

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These results of SAC stability in the plasma fraction of SAC was almost 100% at 3 hr after the addition of SAC to this fraction, however, recovery from the red blood was 87%

at 3 hr [5]. Therefore, high level of SAC in human plasma, might have an important role in the benefits of garlic to health. The S-allyl group in SAC had the highest potency for colon cancer prevention and was almost stable than the other groups, S-methyl, S-propyl and disulfide derivatives [20]. The importance of the S-allyl group on the survival of neurons by using S-allyl group, analogue or derivatives, then only derivatives of S-allyl group were effective [4]. This results indicated that the S-allyl group reacts effective role in disease prevention.

SAC is one of the potential compound obtained from AGE and this compound was increased in the manufacture of garlic. SAC could be used as health claim of biomarker of garlic product that are used neutraceutical and medical ap- plication of human benefits.

Allyl mercaptan

Garlic and garlic compounds are metabolized in the intes- tine and liver, but allicin and some of its transformed con- stituents, such as diallyl disulfide and ajoene, have never been found in the blood, indicating that they are converted to other compounds [11,19]. Some studies have suggested that pure garlic-related organosulfur compounds such as al- licin, ajoene, disulfides and perhaps the trisulfides are me- tabolized primarily to allyl mercaptan shortly after entering the blood [21].

Allicin also has been shown to transform to diallyl sulfide and allyl mercaptan in the isolated perfused rat liver, with the former being rapidly metabolized to allyl mercaptan [8].

However, the physiologic role of allyl mercaptan is well un- known, despite the fact that allyl mercaptan is the major metabolite of garlic compounds. Allyl mercaptan reduces cholesterol synthesis in the rat hepatocytes by the enzymes, 3-hydroxy-3-methyl-glutar-yl coenzyme A (HMG-COA) [18].

Based on well viability, various content ratio up to 100 μg /ml, were chosen as nontoxic levels of allyl mercaptan to carry out subsequent studies.

Allyl mercaptan indicated in a marked inhibition of cho- lesterol in the cells, and also decreased significantly in cho- lesterol secretion [21]. The effecting allyl mercaptan of

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H-acetate into cholesterol with or without 1 ml oleic acid treatment, allyl mercaptan significantly decreased cholester- ol in cells [21,23].

Fig. 5. Effects of various concentrations of allyl mercaptan on cholesterol synthesis in Hep-G2 cells [12]. The cells were incubated in a serum-free Dulbecco's modified Eagles;

medium containing 5, 10, 25, 50, and 100 μg/ml of allyl mercaptan and³H-acetate (1 uci/ml) for 4 hr. Values are mean±SEM (n=6).

Allyl mercaptan treatment of cells incubated with oleic acid indicated in a significantly reduction in the cholesterol synthesis in cells and cholesterol secretion (Fig. 5).

The results is in agreement with the report that allyl mer- captan exerts an inhibitory effect on cholesterol synthesis in rat heptatocytes and inhibition of cholesterol synthesis by allyl mercaptan could be due to the decreased activity of HMG-COA reductase in rat [42]. Although it is rather diffi- cult to interpret the relevance of current data to dietary effect of garlic in vivo, allyl mercaptan, which is a major metabolite of garlic compounds circulating in the blood, could be partly responsible for the cholesterol-lowering effect of garlic. In conclusion, allyl mercaptan is effective in suppressing the synthesis and excretion of cholesterol.

Steroid saponins, non-sulfur compounds

Saponins have characteristic properties, including the pro- duction of a stable foam when shaken with water, a bitter taste, and hemolytic activity. Steroid saponins has been de- tected in AGE. Steroid saponins from garlic led to the iso- lation named sativoside-B

1

, and to the discovery of a known furostanol saponin, proto-desgalactotigonin [18,27]. On the other hand, eruboside-B, a spirostanol saponin correspond- ing to proto-eruboside-B, was isolated from garlic bulbs [15].

Recently the isolation and structure determination of new

steroid saponins named proto-isoeruboside-B and isoerubo-

side-B, which are elucidated to be the C-25 epimers of

pro-eruboside-B and eruboside-B [28]. Steroid saponins and

sapogenins could be considered reliable chemical structure

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biomarkers for the identification of garlic and garlic products. Among the biological activities of steroid saponins islated from the AGE, eruboside-B exhibited antifungal ac- tivity for Candida albicans [16], cardiovascular disease [26], and cytotoxic activities in vitro [28]. Crude saponins from methanol garlic extracts lowered total plasma cholesterol and LDL cholesterol without changing HDL cholesterol lev- els in hypercholesterolemic animal models [40]. Saponins in garlic are reported to exhibit various biological effects, in- cluding cholesterol reduction, and probably effects synerg- istically with organosulfur compounds.

Tetrahydro-β-carboline derivatives

Formation of tetrahydro-β-carboline derivatives (THβCS) were not detect in raw garlic, but they were generated at the beginning of the aging extraction process with intrinsic natural enzyme or high temperature during the aging proc- ess of garlic [12]. These compounds are naturally occurring substances chemically produced during food processing, ex- traction, and storages. THβCS have been identified in soy sauces, beers, wines, chocolate, and cocoa. THβCS are chemi- cally synthesized by condensation between tryptophan and acetaldehyde or pyruvic acid. Acetaldehyde, a precursor of 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCC), is believed to occur from alcohol [12]. On the other hand, in natural aging process of garlic, one is the alliin-alli- cin pathway, which is the most common pathway in garlic, and another is via the Maillard reaction process. Therefore, THβCS were detected in AGE, but not in raw and other proc- essed garlic such as sliced, baked, boiled, crushed, and aging processes. These alkaloids have been demonstrated to ex- hibited antioxidant properties [1], and to inhibit platelet ag- gregation [41], and monoamine oxidase [37], and metal chi- lating, and reducing properties [34], monoamines oxidases [37], monoamine uptake [34], and binding to benzodiazepine receptor [34].

In the present review, AGE was fractionated using hydro- gen peroxide scavenging assay, and THβCS were identified.

THβCS was stronger than common antioxidant, ascorbic acid.

The intracellular level of inducible nitric oxide synthase (INOS) play an important role in determining nitric oxide (NO) production rates in activated macrophages and several other cell types. The high amounts of NO is associated with acute and chronic inflammation and atherosclerosis through cytotoxicity and injury to the surrounding cells and tissues,

THβCS exhibited inhibition at low concentrations. THβCS formed at the beginning of the natural aging process, and the contents increase during the aging. These data suggest that not only organosulfur compounds but also THβCS formed during the natural aging process may contribute to the biological effects of AGE (Fig. 6).

Fructosyl arginine

The amino-carbonyl (Maillard) reaction takes place dur- ing food processing, cooking or storages of food and pro- duced to a complex mixture of reaction products. Maillard reaction products are very heterogeneous and complex, and their exact composition remains mostly unknown. More re- cently, the antioxidant activities of Maillard reaction prod- ucts was shown in vivo study [33]. Feeding rats of Maillard reaction products from arginine, lysine or glycine and sugars significantly inhibited TBARS formation in the liver [24]. It is believed that the observed activities were derived from melanoidine-brown nitrogenous polymer with high molec- ular weight. However the Maillard reaction also produces compounds of low molecular weights fructose-arginine [39].

A low molecular weight Maillard reaction products is lated shown to lower blood pressure and to improve micro- circulation in rabbits. Fructose-arginine, water soluble com- pound, brown color of Maillard reaction product derived from AGE, was used to determine its antioxidant activity toward oxidation of LDL. Fructose-arginine inhibited Cu

^2⁺

induced LDL oxidation. Fructose-arginine on oxidized LDL induced injury in vascular endothelial cells by measuring

Fig. 6. Scavenging effects of tetrahydro-β-carboline derivatives identified in AGE on hydrogen peroxide [23]. Data represent means±SEM of triplicate samples. 1a : (1R,3S)-1- methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid.

1b: (1S, 3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid. 2a: (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β- carboline-1,3-dicarboxylic acid. 2b: (1S,3S)-1-methyl-1,2, 3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid.

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Fig. 7. Effects of fructosyl arginine (Fru-Arg) on oxidized low density lipoprotein (Ox-LDL)-induced lipid peroxidation [16]. Pulmonary arterial endothelial cells were pre- incubated with various concentrations of Fru-Arg at 37^oC and 5% CO2 for 24 hr. Cells were washed with Hanmks' balanced salt solution, incubated with 0.1 mg/ml Ox-LDL for 24 hr, and then thiobarbituric acid reactive substances (TBARS) indicating lipid peroxidation were determined. Data represent means±SE of triplicate samples. Significant difference (P<0.05) compared with control exposed to Ox-LDL but without pre- treatment with Fru-Arg.

lactate dehydrogenase (LDH) release [36]. LDH is an intra- cellular enzyme released into the medium upon cell mem- brane damage. Oxidized LDL caused a significant increase in LDH release (Fig. 7).

There are numerous studies that show Maillard reaction products possess antioxidative activity [27]. The compounds are very important, for the quality of food, because they con- tribute to the development of flavour, color or browning and to changes in the nutritional value [2]. In most cases, Maillard products are tested whereas only in many cases was antioxidative activity related of structurally defined Maillard products [41]. Antioxidative activities of Maillard reaction products have been studied extensively using ami- no acids-sugar as arginine, lysine, glycine or histidine with reducing sugars, have been shown to have very strong bio- logical activities in vitro or in vivo.

Acknowledgement

This research was supported by Kyungsung University Research Grants in 2008.

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